BRPI0209685B1 - insulinotropic peptide derivative and method for producing an insulinotropic peptide derivative - Google Patents

Abstract

"Derivative of an insulinotropic peptide and pharmaceutically acceptable salts produced therefrom, method of producing insulinotropic peptide derivative and use of an insulinotropic peptide derivative and pharmaceutically acceptable salts". The present invention relates to derivatives of an exendin-4 insulinotropic peptide and pharmaceutically acceptable salts thereof, which are useful for the treatment of type ii diabetes. exendin-4 has a chemical structure as shown below: his-gly-glu-gly-thr-phe-thr-ser-asp-leu-ser-lys-gln-x-glu-glu-ala-val- yleu-phe-ile-glu-trp-leu-lys-asn-gly-gly-pro-be-gly-ala-pro-pro-z-where: x represents arg, leu or ile, y represents his, arg or lys and z represents arg-oh, -oh, -nh ~ 2 ~ or lys-oh. The derivatives of the present invention may be prepared by chemical synthetic means and, more easily, by recombinant techniques, which makes commercial production possible.

Description

Field of the Invention [1] The present invention relates to active peptide compounds for the treatment of type II diabetes. [0128] "Insulinotropic peptide derivative and method for the production of an Insulinotropic peptide derivative" In particular, the present invention relates to derivatives of an Exendin-4 insulinotropic peptide.

Background of the Invention [2] It has been found that, with the same amount, oral administration of glucose may stimulate more insulin secretion than injection. After several researches exploring the reason for this, it was found that incretins play important roles in that phenomenon. [3] Incretins are a group of peptide compounds, among which the gastric inhibiting peptide (GIF), which has strong inhibitory activity, and glucagon-like peptide (GLP-1) was described in 1985. There are two types of GLP-1, one is GLP-1 (7-36) amide which is composed of 30 amino acid residues; the other is GLP-1 (7-37), which is composed of 31 amino acid residues. Both have similar activities in stimulating insulin secretion. When acting on pancreatic island cells in vitro, GLP-1 with a concentration as low as 1x10-10 - ΙχΙΟ-llmol / L, are able to stimulate insulin secretion. It was observed that, by DNA analysis and amino acid sequences., These insulinotropic peptides originate from the proteolysis of the proglucagon glucagon precursor under the effect of an intestinal proteolytic enzyme and, therefore, are called peptides similar to glucagon. [4] A feature of GLP-1 is that it can stimulate pancreatic beta cells to synthesize proinsulin and proinsulin mRNA, and to release insulin. The effects of GLP-1 on pancreatic beta cells depend on glucose concentration. When the blood glucose concentration is above 6 mmol / L, GLP-1 significantly stimulates insulin secretion; once concentration is restored to normal level, GLP-1 stimulatory activity can no longer be observed. This feature is widely used in the treatment of type II diabetes. In recent studies, clinical trials among patients with type II diabetes have also verified the effects of GLP-1 on stimulating insulin secretion and decreasing glucose concentration. The chemical structure of GLP-1 (7-36) amide is shown as follows;

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Tyr-Leu-Glu-Gly-Gln-AIa-Ala-Lys ~ Glu-Phe-Ile-Ala-Trp- Leu-Val-Lys-Gly-Arg-NHs 16] in which: (7 J His represents histidine, Ala represents ayanine, Glu represents giutamic acid, Gly represents glycine, Thr represents threonine, Phe represents phenylalanine, Ser represents serine, Asp represents aspartic acid, Val represents valine, Tyr represents tyrosine, Leu represents leucine, Gin represents glutamine, Lys represents lysine, Ile represents isoleucine and ftrg represents arginine. [8] International patent application published under No. WO 91/11457 describes analogues GLP-1 peptides 7-34, 7-35, 7-36 and 7-37, which are useful for the treatment of type II diabetes. [91 An Exendin-4 peptide compound, which was separated from the poison Mexico lizard (Heloderma horridum), was described by Jean-Pierre Ranfman in "Regulatory Peptides" in 1996. Exendin-4 is composed of 39 amino acid residues with the car terminal The chemical structure of Exendin-4 is shown below: 10 His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-20 Lys-Gln-Met-Glu-Glu-Glu- Ala-Val-Arg- 30 Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro 39 Ser-Ser-Gly-Pro-Pro-Ser-NH2 [10] wherein: [11] Met represents methionine, Asm represents, asparagines, and Pro represents praline. [12] Exendin-4 shares 53% homology with GLP-1 in its amino acid sequences. In addition, Exendin-4 has the ability to combine with a GLP-1 receptor and to stimulate insulin secretion. Therefore, it is seen as an insulinotropic peptide. [13] US Patent No. 05424286 describes the results of some comparative experiments conducted between Exendin-4 and GLP-1 with respect to their stimulatory effects on insulin secretion. Compared to GLP-1, Exendin-4 was shown to have a stronger ability to stimulate insulin secretion, and required a lower concentration to exhibit said stimulatory activity. In addition, Exendina-4 has a longer half life in vivo than that of GLP-1. In view of these characteristics, Exendin-4 may become a desirable therapeutic agent for the treatment of type II diabetes. [14] Although Exendin-4 can be prepared by chemical synthesis methods, such as by solid phase synthesis in a peptide synthesizer, the higher cost of this method and the consequent selling price, removes Exendin-4 from commercialization. Thus, for the purpose of commercialization, bioengineering techniques were attempted to try to reduce the cost of production.

Objectives of the Invention [15] The present invention is directed to providing some derivatives of an Exendin-4 insulinotropic peptide. Said derivatives help stimulate insulin secretion and reduce blood glucose concentration. Therefore, they can be used for the treatment of type II diabetes. Said derivatives may be prepared by synthetic chemical means, and more easily by recombinant techniques, which make it possible to reduce production costs. Summary of the Invention [16] The present invention provides derivatives of an insulinotropic peptide and pharmaceutically acceptable salts produced thereof. Said insulinotropic peptide is provided with the amino acid sequence shown below: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu 14 Ser-Lys-Gln-X-Glu-Glu-Glu -Ala-Val-Y- 30 Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-39 40 Pro-Be-Gly-Wing-Pro-Pro-Pro-Ser-Z [ 17] wherein: [18] X represents Arg, Leu, Ile or Met; Y represents HIs, Arg or Lys; 2 represents Arg-OH, -OH, -NH 2 or Lys-OH; and [19J when X is Met and Y is Arg, Z cannot be NH2. [20] The insulinotropic peptide derivatives of the present invention have an acidic amino acid sequence as shown in the Sequence Listing, in particular as described in <210> - <21G> 4. [21] Insulinotropic peptide derivatives of the present invention are amphoteric compounds, and may be sufficiently acidic or sufficiently basic to react with any number of inorganic bases and organic or inorganic acids to form a salt. The acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, roetanesulfonic acid, oxalic acid. , p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of said salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, phosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, propanoate, caproate , oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butino-1,4-dioate, hexine-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate phenylpropionate, phenylbutyrate, citrate, lactate, gamma hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and in particular hydrochloric acid. [22] Alkaline may also be employed to react with derivatives of the present invention to form salts. Representative examples of said alkali include ammonia, alkali metals, alkali metal hydroxides, carbonates and bicarbonates. Typically, said alkaline may be sodium hydroxide, potassium hydroxide, ammonium hydroxide and potassium carbonate. [23] One aspect of the present invention provides a method for producing a solid phase synthesis insulinotropic peptide derivative comprising the use of HMP resin as a solid phase carrier which protects alpha-alanine from an amino acid with 9-fluorenyl methoxycarbonyl (Fmoc), which synthesizes the residue into a synthesizer peptide by the amino acid sequence of an insulinotropic peptide derivative, and the products are obtained after the separation, purification and lyophilization steps. In another aspect, the present invention provides a method of producing insulinotropic peptide derivatives by recombinant techniques comprising: synthesizing gene fragments by the amino acid sequence of an insulinotropic peptide derivative. , [26] B. obtain a cloned bacterial strain after ligation with gene fragments, construction of a recombinant plasmid, bacterial cell culture and recombinant plasmid transformation, [27] C. extract inclusion bodies after fermentation of the bacterial strain. and cell wall collapse, [28] D. Obtaining the final product by inclusion body lysis, crude product separation, HPLC purification and lyophilization step. [29] In yet another aspect, the present invention provides use of said insulinotropic peptide derivatives and the pharmaceutically acceptable salts thereof in a type II diabetes treatment. [30] The present invention proposes novel derivatives of the Exendin-4 peptide compound, which is in the range of Exendin-4 derivatives. The derivatives of the present invention exhibit biological activities of stimulating insulin secretion and reducing blood glucose concentration. They can be used for the treatment of type II diabetes mellitus. Said derivatives may be prepared by chemical synthesis and more easily by recombinant techniques, which make it possible to reduce production costs. In addition, the biological activities of these derivatives have been proven by pharmacodynamic studies among non-obese diabetic mice (NOD), when 0.1 Gg of derivatives of the present invention were injected abdominally into the mouse, insulinotropic peptide derivatives had obvious effects in increasing insulin secretion and reduce the blood glucose level. Description of the Figures [31] The present description will now be further illustrated with reference to the following Figures and examples. [32] Figure 1 shows the results of pharmacodynamic studies for insulinotropic peptide derivatives of the present invention. [33] Figure 2 shows the results of mass spectrum analysis for the product prepared in Example 1, with its theoretical molecular weight of 4300.6 and its determined molecular weight of 4316.7. [34] Figure 3 schematically illustrates the experimental results of the decreasing blood glucose effects of Exendin 4 (Lys20, Arg40) and Exendin 4 (Leul4, Lys20, Arg40). [35J Figure 4 schematically illustrates the results of the experiment on the stimulatory effect of Exendin 4 (Lys2Ó, Arg40] on insulin secretion. [36J Figure 5 schematically illustrates the results of the stimulatory effect experiment on peptide secretion. -C for Exendin 4 [iys20, Arg40], Description of the Invention [37] The following examples are illustrative and are not intended to limit the scope of the present invention in any way Example 1 [38] Preparation of an insulinotropic peptide derivative where X is Arg , Y is Lys, and Z is -OH by solid phase synthesis. (1) Amino acid monomers [39] wherein: [40] Fmoc represents 9-fluorophenyl methoxycarbonyl [41] BOC represents tert-butyloxycarbonyl [42] Trt represents trityl [43] OtBu represents tertiary butyl ester, and [44] tBu represents tert-butyl. (2) Apparatus and Reagents [45] Apparatus: Peptide Synthesizer Model 433A (Applied Biosystem, US) [46] Reagents: N -methyl ketopyrrolidine, c methylene chloride, hexahydropyridine, methanol, dimethylaminopyridine, DMF N, N-diisopropylethylamine / NMP, 100 mmol HBTU / 0.5 Μ HOBT in DMF, N, N-dicyclohexylcarbodiimide / NMP [47] In which: [48] DMF represents Ν , Β-dimethylformamide [49] NMP represents N-methylpyrrolidone [50] HOBT represents 1-Hydroxybenzotriazole, and [51] HBTU represents 2- (1H-benzotriazola-yl-1,1,3,3-tetramethyluronium) hexafluorophosphate . (3) Procedures: a. Synthesis [52] Considering the synthesis scale of 0.25 for example, the synthesis process was described as follows. 0.25 g of HMP resin was weighed and disposed in a reactor container of the entire synthesizer. 1 mmol of various residues, each of which coupled with protecting groups, were weighed and structured on the synthesizer by the amino acid sequence of the insulinotropic peptide derivative from the carboxy terminal to the amino terminal. At room temperature of 25 ° C, the Fmoc protection removal reactions, which activate the residue and fix the activated residue to the HMP resin, were automatically performed under the control of a computer program. Said reactions were circulated until the entire peptide was synthesized. Upon completion of the synthesis, the resin fixed to the residue, with each residue coupled with the side chain protecting groups, was air dried on a peptide synthesizer and then weighed. B. Removal of the protecting groups and removal of the resin: [53J The resin fixed to the residue, with each residue of the insulinotropic peptide derivative coupled with the protecting groups, was disposed in a capped Erlen Meyer flask ·, followed by the addition of reagent reagents. divagem shown as below. [54] The electromagnetic stirring reaction was performed at a constant temperature of 30 ° C for 6 hours. After the filtration step, the aqueous filtrate was collected. The resin was washed with small amount of trifluoroacetic acid. Then the collected aqueous filtrate and wash solution were mixed together, and ether was added to the precipitation. The mixture was filtered, and the resulting precipitate was washed with a small amount of ether. After evaporation in a dehumidifier, the crude product was obtained. ç. HPLC Purification and Lyophilization [55] Separation and purification of the crude product was achieved using preparative HPLC. The final product was obtained after the freezing and freeze drying step. The molecular weight of the product was determined using spectrogram junction analysis and mass chromatography. The peptide derivative was found to have a molecular weight of * 3316.7, the theoretical molecular weight of which was 4300.6.

Example 2 Preparation of an insulinotropic peptide derivative where X is Leu, Y is Lys and 2 is Arg-OH by bioengineering techniques. [57] Synthesization of gene fragments by amino acid sequence of insulinotropic peptide derivative (1) 5'AAT TCC ATG CAC GGC GAA GGC ACC TTC ACC AGC

GAT CTG AGC AAA CAG CTG GAA GAA GAA GCG GTT AA

(2) 5 * ACTG TTC ATC GAA TGG CTG AAA AAC GGC GGC CCG

AGC AGC GGC GCG CCG CCG CCG AGC CGT TAG A

(3) 5 'AGCTT CTA ACG GCT GCC GCC GCC GCC GCC GCC GCT GCT GCC GCC GCC GTT TTT CAG CCA TTC GAT GA

(4) 5'AC AG TTT AAC CGC TTC TTC TTC CAG CTG TTT GCT CAG ATC GCT GGT GAA GGT GCC TTC GCC GTG CAT B. Cloning Binding: [58] Two tubes were obtained, and fragment (1) and fragment ( 4) with their OD260 nm = 0.1 [optical density at 260 nm) were placed in one tube, while fragment (1) and fragment (4) with the same optical density were placed in another tube. Then, polynucleotide kinase buffer, polynucleotide kinase and ATPs were added to the two tubes respectively. The reaction mixtures were incubated at 37 ° C for 60 minutes to constitute the 5 '' terminal of the phosphorylated gene fragment. Then the two tubes were placed in a 95 ° C water bath and incubated for 10 minutes. Heating of the water bath was stopped and was naturally cooled to room temperature while the annealing reaction was carried out during the process. T4 ligase and T4 ligase buffer were added to the two tubes respectively, and the mixtures were incubated overnight at 16 ° C for ligation of gene fragments.

Plasmid [59] A plasmid containing such a Lac, PL or Tac promoter was placed in a tube and digested with restriction endonucleases EcoRI and HindIII. The digested plasmid was extracted with hydroxybenzene: chloroform solvent and collected by centrifugation. The aqueous phase was maintained and washed three times with chloroform solvent. Centrifugation was continued, and the resulting aqueous phase was precipitated with isopropanol solvent, followed by centrifugation and air drying step. [60] The digested plasmid and ligated fragment were mixed together. T4 ligase and T4 ligase buffer were added to the mixture. The ligation reaction was performed at room temperature for 3-4 hours.

Host bacterial cell culture: [61] E. coli JM 103 bacterial cells were incubated with shaking at 37 ° C in LB liquid medium (1000 ml LB liquid medium containing 10 g peptone, 5 g yeast extracts and 5 g of sodium chloride) for 4 hours. Afterwards, the bacterial cultures were centrifuged, the collected bacterial cells were treated with calcium chloride solution and kept at -4 ° C for later use.

Transformation [62] The cloned plasmid was transformed into. E. coli IM 103 host cells. Transformed bacterial cells were incubated in an ice bath for 30 minutes, and then incubated at 42 ° C for 2 minutes. Bacterial cells were spread on an antibiotic ampicillin-containing agar plate, and incubated overnight at 37 ° C. Screening of the colonies was conducted later, and colonies containing the recombinant plasmid were maintained as positive colonies. [63] C. Fermentation [64] A selected colony that was endowed with a recombinant plasmid carrying the derived gene was incubated with shaking LB liquid medium. 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG) was added for the purpose of protein induction. Bacterial cells were incubated overnight and collected by centrifugation. The expressed protein was identified by polyacrylamide gel electrophoresis (PAGE) containing 12% sodium dodecanesulfonate. D. Inclusion Bodies [65] Ten bottles, each containing 300 ml of bacterial cultures, were incubated with shaking under the conditions mentioned above. After the protein incubation step, lysis solution (20 mM phosphoric acid buffer containing 1% sodium chloride, pH 7.5) and lysozyme were added. Bacterial cultures were incubated at 30 ° C for 30 minutes and centrifugation was followed. The collected precipitate was treated with 6M guanidine hydrochloride (Gu.HCl) for extraction of inclusion bodies. Centrifugation was continued and the resulting supernatant was dialyzed to remove guanidine hydrochloride. The dialysate was washed three times with 20 mM phosphonic acid buffer (containing 1% sodium chloride and 0.1% Tween 80, pH 7.5), and the inclusion bodies were obtained later. E. Lysis [66] Inclusion bodies were dissolved in 8M carbamide solution. Hydrochloric acid was added at a concentration of 50 mM. After the addition of cyanogen bromide, the solution was stirred under light shielding and under nitrogen shielding. The lysis reaction was conducted for 2 hours, followed by HPLC analysis. F. Purification [67] Upon completion of the lysis reaction, the crude product was obtained by chromatography division on Sephadex G-25 and the final product was purchased by HPLC purification. As with the result of solid phase synthesis, it was shown by mass spectrographic analysis that the determined molecular weight of the peptide derivative is consistent with the theoretical weight.

Example 3 Pharmacodynamic Studies [68] Studies were conducted in two-hour fasting non-obese diabetic mice (NOD mice) weighing 17 ± 2 kg. Mice in control group 1 received a 2 Dg abdominal injection of insulin, and mice in control group 2 received a 4 Dg abdominal injection of insulin, while mice in the experimental group received a 0.1 Dg abdominal injection of insulin. insulinotropic peptide derivative obtained as described in Example 1. [69] Blood samples were collected at different minutes. Plasma glucose concentrations were determined using a plasma glucose test kit (Shanghai Institute of Biological Products Ministry of Health). The results were shown in the Figures. It can be seen that the decreasing effect of insulinotropic peptide derivative on blood glucose is relatively obvious.

Example 4 [70] The decreasing effects of Exendin 4 (Lys20, Arg40) and Exendin 4 (Leul4, Lys20, Arg40) on blood glucose in NOD mice.

Materials and Methods: [71] NOD mice were provided by Shanghai Animal Laboratory Center of Chinese Academy of Sciences. 0.9% sodium chloride solution, Exendin 4 (Lys20, Arg40) and Exendin 4 (Leul4, Lys20, Arg40) peptides were used in the test. The plasma glucose test kit was purchased by Shanghai Institute of Biological Products Ministry of Health. [72] Overnight fasting NOD mice were divided into three groups. Mice from the first group were injected abdominally with 200 Dl of solution containing 40% glucose and 1 kg of Exendin 4 (Lys20, Arg40). Mice from the second group were injected abdominally with 200 Dl of solution containing 40% glucose and 2 kg of Exendin 4 (Leul4, Lys20, Arg40). While in the third group, also the control group, mice were injected abdominally with the glucose solution. [73] 30 G1 blood samples were obtained from the retroorbital sinus zeinous, immediately using a calibrated capillary that was previously treated with heparin. The sample was placed in 300 µl normal saline and mixed with the saline. Erythrocytes were removed by centrifugation at 3000 rpm, while blood serum was maintained for glucose determination. Different blood samples were obtained as described above at 30 minutes, 60 minutes and 120 minutes, respectively, then the blood sera were separated. Glucose concentrations of the three plasma samples were determined according to the method described by the test kit, and the effect of GLP-1 (7-36) on blood glucose concentration was detected. [74] As shown in Figure 1, after a dramatic increase, the blood glucose concentration in the control group gradually returned to a normal level; whereas glucose concentrations in the experimental groups never showed a noticeable increase and maintained a normal level all the time. The reason is that administration of Exendin 4 derivative stimulates insulin secretion and thus avoids the dramatic fluctuation of glucose concentration. Thus, the reducing effects of Exendin 4 (Lys20, Arg40) and Exendin 4 (Leul4, Lys20, Arg40) on blood glucose are supported by the results of the experiment.

Example 5 Stimulatory Effect of Exendin 4 (Lys20, Arg20) on Insulin Secretion Materials and Methods [75] NOD mice were provided by the Shanghai Laboratory Animal Center of Chinese Academy of Sciences. 40% glucose solution, 0.9% sodium chloride solution and Exendin 4 (Lys20, Arg40) were used in the insulin radio immunoassay kit, was purchased by Shanghai Institute of Biological Products Ministry of Health. [76] NOD mice were divided into two groups. 50 Gl of blood sample was obtained from the venous plexus of the eye using a calibrated capillary, the inner wall of which was rinsed with 1 mg / ml heparin and was previously air dried. Mice in both groups were injected abdominally with 200 Gl of normal saline containing 5 Gg of Exendin 4 (Lys20, Arg40), respectively, and this time was recorded as 0 min. Different blood samples were obtained as described above at 5 min, 10 min, 20 min and 30 min, respectively. After sampling, each blood sample was immediately placed in a centrifuge tube containing 50 Dl of normal saline and mixed with saline. Erythrocytes were removed by centrifugation at 3000 rpm. Insulin concentrations of the different samples were determined by following the methods described by the radio immunotest kit, and then the stimulatory effect of Exendin 4 (Lys20, Arg40) on insulin secretion was detected. [77] As shown in Figure 2, abdominal injection with Exendin 4 (Lys20, Arg40) can significantly stimulate insulin secretion.

Example 6 Stimulatory Effect of Exendin 4 (Lys2of Arg40) on C-Peptide Secretion

Materials and Methods [78] Healthy C57 / BL mice were provided by the Shanghai Animal Laboratory Center of Chinese Academy of Sciences. 40% glucose solution, 0.9% sodium chloride solution and Exendin 4 (Lys20, Arg40) were used in the experiment. The insulin radioimmuno test kit, and C-peptide radio immuno test kit were purchased from the Shanghai Institute of Biological Products Ministry of Health. [79] C57 / BL mice were divided into two groups. 50 Dl of blood sample was obtained from the venous plexus of the eye using a calibrated capillary, the inner wall of which was rinsed with 1 mg / ml heparin and was previously air dried. Mice in both groups were injected abdominally with 200 01 normal saline containing 5 Dg Exendin 4 (Lys20, Arg40), respectively, and this time was recorded as 0 min. Different blood samples were obtained as described above at 5 min, 10 min, 20 min and 30 min, respectively. After sampling, each blood sample was immediately placed in a centrifuge tube containing 50 Dl of normal saline and mixed with saline. Erythrocytes were removed by centrifugation at 3000 rpm. The C-peptide concentrations of the different samples were determined by following the methods described by the radio immunotest kit, and then the stimulatory effect of Exendin 4 (Lys20, Arg40) on C-peptide secretion was detected. [80] As shown in Figure 3, abdominal injection with Exendin 4 (Lys20, Arg40) can significantly stimulate C-peptide secretion.

Claims (4)

1. Derivative of insulinotropic peptide, and pharmaceutically acceptable salts produced therefrom, characterized by the fact that said derivative has the following amino acid sequence: His-Gly-Glu-Gly-Thr-phe-Thr "Ser" Asp "Leu" Ser-Lys-Gln-X-Glu-Glu-Glu-Ala-Val-Y-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn ^ Gly-Gly-Pro-Be-Ser-Gly-Ala- Pro-Pro-Pro-er-2, where X represents Leu, Y represents Lys, and 2 represents Arg-QH. Insulinotropic peptide derivative according to claim 1, characterized in that said derivative has the amino acid sequence below: Gx-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser- Lys-Gln-X-Glu-GIu-Glu-Ala-Val-Y-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Fro-Be-Gly-Ala-Pro- Pro-Pro-Ser-Z, where X represents Met, Y represents Lys, and 2 represents Arg-QH » A method for producing an insulinotropic peptide derivative as described in claim 1 or 2 by solid phase synthesis, which comprises the use of an HMP resin as a solid phase carrier, the protection of alpha- amino acid of an amino acid with 9-fluorenyl methoxyearfaoniia (Fmoc), the synthesis of a residue on a peptide synthesizer by the amino acid sequence of a peptide insulinotropic derivative, and obtaining products after the separation, purification and lyophilization steps . Method of producing an insulinotropic peptide derivative according to claim 3, characterized in that it comprises: (a) synthesizing gene fragments by the amino acid sequence of the insulinotropic peptide derivative, (b) obtaining of a cloned bacterial strain after ligation of the gene fragments, construction of a recombinant plasmid, bacterial cell culture and transformation of the recombinant plasmid, (c) extraction of inclusion bodies after fermentation of the bacterial strain and (d) obtaining the final product after inclusion body lysis, crude product separation, HPLC purification and lyophilization step.